Inhibition of cannabinoid degradation enhances hippocampal contextual fear memory and exhibits anxiolytic effects

Summary Recent studies have demonstrated the pivotal involvement of endocannabinoids in regulating learning and memory, but the conclusions obtained from different paradigms or contexts are somewhat controversial, and the underlying mechanisms remain largely elusive. Here, we show that JZL195, a dual inhibitor of fatty acid amide hydrolase and monoacylglycerol lipase, can enhance the performance of mice in a contextual fear conditioning task and increase the time spent in open arms in the elevated zero maze (EZM). Although the effect of JZL195 on fear memory could not be inhibited by antagonists of cannabinoid receptors, the effect on the EZM seems to be mediated by CB1R. Simultaneously, hippocampal neurons are hyperactive, and theta oscillation power is significantly increased during the critical period of memory consolidation upon treatment with JZL195. These results suggest the feasibility of targeting the endocannabinoid system for the treatment of various mental disorders.


INTRODUCTION
Memory deficits are prominent features of many mental disorders, such as anxiety, 1 insomnia, 2 and even Alzheimer's disease (AD). 3In recent decades, the development of various drugs that were anticipated to alleviate memory deficits but that eventually showed limited clinical benefits has been observed. 3As a widely distributed neuromodulatory system, the endocannabinoid system (ECS) and its relative chemical compounds are involved in virtually all brain functions through either traditional cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) or other G protein-coupled receptors (GPRs). 4Retrograde control of synaptic transmission and plasticity is a powerful way through which the ECS regulates brain functions; 5 hence, the ECS may serve as a therapeutic target in neurological disorders.CB1R is the key mediator of endocannabinoid-elicited short-and long-term synaptic plasticity, which is regarded as the neurological basis of learning and memory, in the hippocampus, prefrontal cortex, and amygdala. 6,7Although sparsely expressed in the central nervous system (CNS), CB2Rs are also indispensable for memory recall. 8Traditional cannabinoids (e.g., D 9 -tetrahydrocannabinol, THC) have been demonstrated to trigger memory impairment 9 and an increased incidence of mental diseases because of their psychotropic properties. 10In this regard, cannabidiol (CBD), a nonpsychotropic Cannabis-derived compound, has been found to improve the performance of mice in memory-retrieval tasks 11,12 and has been applied in registered clinical trials for several diseases. 10,13he concentrations of endocannabinoids are collaboratively controlled by enzymes that catalyze the synthesis and hydrolysis of these metabolites.In particular, the hydrolysis of endocannabinoids, 2-arachidonoyl glycerol (2-AG) and anandamide (AEA), is catalyzed preferably by monoacylglycerol lipase (MAGL) and fatty acid aminohydrolase (FAAH), respectively. 14Generally, inhibition of MAGL and FAAH causes an accumulation of 2-AG and AEA within synapses, leading to the reinforcement of cannabinoid-induced intracellular signaling and synaptic plasticity.Global MAGL knockout promotes hippocampus-dependent spatial memory recall in mice in a water maze test. 15However, dual ablation of MAGL and FAAH through pharmacological inhibition or genetic manipulation results in a THC-like detrimental effect on memory, as evaluated by the performance of mice in the water maze task. 16][19] To pharmacologically inhibit MAGL and FAAH individually or in combination, JZL184 (target to MAGL), URB597 (or PF-3845, target to FAAH), and JZL195 (target to both MAGL and FAAH) are often used.JZL184 typically inhibits MAGL and then results in an increased level of 2-AG, but at a high dose (40 mg/kg) it may also inhibit FAAH. 16In contrast to JZL184 and URB597, JZL195 seems to work in a more complex way.JZL195 has also been demonstrated to modulate neuroinflammation, 20 locomotion, 21 pain, 22 and self-grooming 23 through CB1Rs across species under certain circumstances.Although the role of the single drugs JZL184 and URB597 in fear conditioning and extinction has been investigated, 24 we failed to search the literature in PubMed about JZL195 when using keywords such as ''JZL195,'' ''fear,'' and ''fear memory,'' alone or combined.Therefore, showing the effect and underlying mechanism of JZL195 on fear memory, in terms of its potential as a clinical drug when targeting various mental disorders, is essential.
Due to these inconsistent results, it is of great necessity to probe the role of JZL195 in contextual fear memory formation and anxiety-like behavior.To address these issues, we used fear conditioning and the elevated zero maze (EZM) to assess fear memory and anxiety-like behavior, immunofluorescence and electrophysiology to assess neuronal activity, and pharmacology and trans-genetic mouse lines to assess the role of CB1R and CB2R.Overall, we found that the administration of JZL195, a dual inhibitor of FAAH and MAGL, could promote contextual fear memory consolidation and recall and exhibited anxiolytic effects.

Inhibition of cannabinoid hydrolysis was anxiolytic and enhanced contextual but not cued fear memory
Fear memory has been studied in both cue-dependent and context-dependent fear conditioning paradigms.We found that systemic administration of JZL195 did not change the basal freezing time of mice in the absence of foot shocks in contextual conditioning recall (Figures 1A  and 1B) and also the intact short-term memory (Figure S1).Then, we employed a contextual fear conditioning paradigm for the test.Compared with that of untreated mice, those treated with JZL195 displayed significantly prolonged freezing time on Day 2 in the recent recall test, and this increasing effect was abolished in the remote recall test (Figure 1D).Next, we explored the effect of JZL195 on cue fear conditioning paradigms.However, we failed to detect a significant difference between vehicle-and JZL195-treated mice in the cued fear memory recall test (Figures 1E-1G) or basal contextual freezing in Context B (Figure 1F).Mice also spent much more time in the open arms of the EZM (Figure 2), with no modification of traveling distance (Figure 2B).These results showed that JZL195 promotes contextual fear memory consolidation and recall but not cued fear memory.

JZL195 enhanced c-Fos expression induced by the contextual fear memory test
We then tested whether increased freezing could activate the hippocampus, which plays a critical role in contextual fear conditioning.The procedure of the experiment is shown in Figure 3A, and a representative image of c-Fos expression is shown in Figure 3B (Left: Cornu ammonis 1 (CA1), Middle: Cornu ammonis 3 (CA3), Right: Dentate gyrus (DG)).The number of c-Fos-positive cells was higher in the hippocampal CA1 region of JZL195-treated mice than in vehicle-treated mice (Figure 3D).The hyperactivity of neurons was also found in the CA3 (Figure 3E) but not the DG (Figure 3F) region.Correlation analysis also showed that freezing time had a positive correlation with the number of c-Fos + cells in CA1 (Figure 3G) and CA3 (Figure 3H) but not in the DG (Figure 3I).These data showed that the improvement in contextual fear memory consolidation and recall induced by JZL195 was accompanied by an increase in neuronal activation in the hippocampus.

JZL195 enhanced hippocampal theta oscillation power in CA1 and neuronal activity
Two hours after drug injection, the local field potential (LFP) data revealed a significant enhancement in the relative power of theta oscillations in CA1 of JZL195-treated mice (Figure 4D).Meanwhile, the single-unit activity measurement did not show any difference compared to the baseline (Figure 4C).Twenty-four hours later, a statistically significant increase in single-unit activity was measured in JZL195-treated mice (Figures 4A-4C).Contextual fear conditioning was then introduced.Although mice from both groups showed similar CA1 neuronal activity, the firing rate of all isolated single units recorded in JZL195-treated mice was significantly increased (Figures 4E-4G).These data collectively suggested that enhanced activities in the hippocampus were induced by JZL195.

Excitatory synaptic transmission was changed in CA1 pyramidal neurons
Miniature excitatory postsynaptic current (mEPSC) was thus recorded in the CA1 pyramidal neurons (Figure 5A).Compared to those from vehicle-treated mice, neurons from JZL195-treated mice exhibited a decreased frequency of mEPSCs (Figures 5B and 5C) and an elevated average amplitude (Figures 5D and 5E).These results indicated that the intrahippocampal circuit may contribute to the increased neuronal activity in the CA1 area after JZL195 treatment.

Hippocampus-specific knockout of CB1R blocked the anxiolytic effect of JZL195 but not fear memory recall
As shown in Figures S2A and S2B, intraperitoneal injection at doses of 0.03 and 3 mg/kg did not influence the performance of mice in the contextual fear conditioning task.Neither AM281 and NESS0327 nor SR144528 could counteract the effect of JZL195 (Figures S3A-S3D).Although increased freezing time was observed in all of the JZL195-treated mice, it did not tend to be decreased by the coinjection of the antagonist of CB1R or CB2R.As shown in Figure 6, the CB1R protein could not be detected in the hippocampus only, while CB1R expression in the other brain areas was not affected.The timeline of this experiment is shown in Figure 7A.As shown in Figure 7, wild-type mice spent more time in the open arms of the EZM 24 h after the drug injection test (Figure 7B), while CB1R HÀKO mice spent even less time in the open arms.We did not observe any difference in the distance traveled or the frequency of entering the open arms between these two mouse lines (Figures 7A and 7C).CB1R HÀKO mice showed freezing time comparable to that of wild-type mice during contextual fear conditioning (Figure 7F) and the short-term memory test (Test 1, Figure 7G).On the other hand, both wild-type mice and CB1R HÀKO mice had longer freezing times in the recent memory test (Test 2, Figure 7H).These results suggested that JZL195 may contribute to memory consolidation and recall in a noncannabinoid receptor-dependent manner, but hippocampal CB1R may mediate the anxiolytic effect induced by JZL195.

DISCUSSION
Endocannabinoids have been well documented to play crucial roles in learning and memory. 14,25In the literature, controversial results regarding the intervention effect of endocannabinoid-degrading enzymes have been collected thus far.Knockout of MAGL, an enzyme that predominantly degrades 2-AG, was reported to promote spatial learning and memory in the water maze task and the long-term plasticity of CA3-CA1 synaptic transmission. 15However, in another report, both JZL195 and JZL184 (a selective inhibitor of MAGL) were observed to impair the performance of mice in the water maze. 16he influences of MAGL and FAAH deficits on fear conditioning also seem inconsistent across studies.Direct injection of AEA into the nucleus accumbens (NAc) core slightly reduced the freezing time during the contextual fear memory retrieval task, 26 and systemic administration of URB597, a selective inhibitor of FAAH, impaired fear memory recall. 27However, when the time point of drug injection was moved backward to the start of the learning phase, the systemic administration of URB597 enhanced contextual fear memory acquisition, and this effect can be inhibited by JZL185 delivered into the prefrontal cortex or the ventral hippocampus. 28If the clue was set as a tone, the systemic administration of JZL184 did not affect fear conditioning, but the fear memory was too strong to be extinguished. 24In addition, the enhancement of tone-cued fear memory recall induced by JZL184 but not URB597 was also reported. 29Despite these different reports, all the aforementioned positive outcomes achieved from drug administration or genetic manipulation are CB1R dependent.Few studies have been designed to probe the potential role of CB2R, although it was still reported as an important player in contextual fear memory 8 and a key mediator of JZL184's effects on fear memory. 29In the present study, we found that JZL195, a dual inhibitor of MAGL and FAAH, enhanced the performance of mice in the contextual memory retrieval task.This effect disappeared 30 days later when the mice were returned to Context A as a remote memory test.We also found that the dual inhibition of MAGL and FAAH did not affect the consolidation and recall of tone-cued fear conditioning, as mice did not show much more freezing time in Context B in the presence of training tone.This result suggested that JZL195 may take effect in a context-related brain area such as the hippocampus, which has been identified as a crucial brain area taking part in the regulation of contextual memory and fear memory, as evidenced by significantly increased c-Fos-expressing neurons after contextual fear memory recall.1][32][33] We found here that the theta oscillation power was higher than the basal level 2 h after JZL195 injection.Since the critical period of memory consolidation after acquisition ranges from 2 to 6 h, 34 the increase in theta oscillation in our study may suggest that JZL195 has the potential to promote memory consolidation.
In this study, a decreased frequency of mEPSCs was observed, which indicated a presynaptic change triggered by JZL195.Conversely, the increased amplitude of mEPSCs is often regarded as a postsynaptic change resulting from upregulated glutamate receptor subunits, mainly a-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR), and enhanced density of dendritic spines.All these alterations may contribute to high levels of neuronal activity and potential long-term potential (LTP) that underlie efficient memory retrieval.The effect of endocannabinoid (eCB) related drugs is often considered to work through CB1R or CB2R and therefore the following intracellular pathways.However, in the present study, we did not succeed in illustrating the necessity of either CB1R or CB2R for the effect of JZL195 because both the antagonists and knockout mice failed to inhibit the increased freezing.This result suggests that the other newly found receptors (e.g., TRPV1 and GPR55) may take the place of CB1R and CB2R in the contextual fear memory task.
In addition to contextual fear memory, we also found that the injection of JZL195 at a dose of 20 mg/kg produced an anxiolytic effect in the EZM test, which was mediated by CB1R, and this effect was abolished by genetic hippocampus-specific deletion of CB1R.The potential therapeutic effect of eCB has been discussed for decades; however, the dose and the injection time point do matter when applying eCB-related reagents such as JZL184, URB597, PF-3845, and JZL195.For JZL195 specifically, 3 mg/kg administration produced an antidepressant effect in an major drepressive disorder model 24 h after the last injection 35 but was reported as pro-depression when directly introducing mice into the force-swimming test 3 h after injection, 36 as was 20 mg/kg in the same report.In an anxiety-like behavioral test, when JZL195 was administered at 10 mg/kg and 2 h before the test, mice showed no such alteration in anxiety state either following acute stress or not. 37JZL184 exhibited anxiolytic effects at various doses 3 h after injection. 38Consistent with one of the aforementioned reports, 35 we found in this study that JZL195 produced an anxiolytic effect at a dose of 20 mg/kg when applied 24 h before the EZM test.This result also contradicts some other reports that showed the complex effect and underlying mechanism of JZL195.When CB1 HÀKO mice were involved, JZL195 even had a pro-anxiety effect.A possible mechanism is that the anxiolytic effect was mediated by CB1Rs expressed on glutamatergic axon terminals, 39 as we revealed a decrease in the frequency of mEPSCs.Once CB1R is removed, accumulated eCB, particularly AEA, activates TRPV1 receptors, which then increase anxiety-like behavior. 40n summary, we established that JZL195 significantly promotes memory consolidation and retrieval in a foot shock-based fear memory task.However, due to the discrepancies among behavioral test paradigms or other unknown reasons, including pharmacological and multi-brain area interaction factors, further investigations are probably needed before the precise and detailed role that the ECS plays in different phases of memory formation can be elucidated.Moreover, we also reported a hippocampal CB1R-dependent anxiolytic effect of JZL195.Nonetheless, our study suggests the feasibility of targeting endocannabinoid-degrading enzymes for the clinical treatment of mental disorders.

Limitations of the study
In this study, we found that the dual inhibitor of FAAH and MAGL could enhance the performance of mice in the contextual fear memory task and the activity of the hippocampus.This is the first time that JZL195 was found to benefit a typical type of memory at a high dose.Although this study suggests the possibility of JZL195 contributing to the treatment of mental disorders, more specific experiments involving disease models should be conducted to identify the potential of JZL195.The subsequent mechanism underlying the anxiolytic effect of JZL195 should be investigated in detail.

Figure 3 .
Figure 3. c-Fos expression in the hippocampus was increased after the contextual test (A) Immunofluorescence experiment procedure.(B) Representative images of c-Fos expression in CA1 (Left), CA3 (Middle), and DG (Right).(C) The freezing time of mice that were used to detect c-Fos expression in the recent test (unpaired t test, t (5) = 2.980, p = 0.031).(D) The number of c-Fos-positive cells in the CA1 area of the hippocampus (unpaired t test, t (37) = 4.800, p < 0.0001).(E) The number of c-Fos-positive cells in the CA3 area of the hippocampus (unpaired t test, t (37) = 3.564, p = 0.001).(F) The number of c-Fos-positive cells in the DG area of the hippocampus (unpaired t test, t (37) = 0.953, p = 0.347).(G) Correlation analysis between the number of c-Fos-positive cells in the CA1 area of the hippocampus and freezing time (F test, F (1,5) = 6.440, p = 0.052).(H) Correlation analysis between the number of c-Fos-positive cells in the CA3 area of the hippocampus and freezing time (F test, F (1,5) = 23.860,p = 0.005).(I) Correlation analysis between the number of c-Fos-positive cells in the DG area of the hippocampus and freezing time (F test, F (1,5) = 0.067, p = 0.807).n = 22 slices from 4 vehicle-treated mice and n = 18 slices from 3 JZL195-treated mice.Data are represented as the mean G SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 6 .
Figure 6.Construction and verification of the hippocampus-specific deletion of CB1R (A) The scheme of the virus injection (Left) and the timeline of the experiment (Right).(B) Representative image of the CB1R-stained hippocampus from wild-type mice (scale bar: 200 mm).(C) Representative image of the CB1R-stained hippocampus from CB1R HÀKO mice (scale bar: 200 mm).(D) Enlarged representative image of the CB1R-stained hippocampus from wild-type mice (scale bar: 20 mm).(E) Enlarged representative image of the CB1R-stained hippocampus from CB1R HÀKO mice (scale bar: 20 mm).